Method for renal disease diagnosis and prognosis using annexin A1 and Rab23 as markers

ABSTRACT

Use of Annexin A1 or Rab23 as a biomarker for diagnosing kidney disease or assessing efficacy of kidney disease treatment.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to Taiwanese Patent Application098104215, filed Feb. 10, 2009, the content of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Renal disease affects millions of people each year. Early diagnosisgreatly improves efficacy of renal disease treatment. Thus, there is aneed for developing an accurate method for diagnosing renal disease.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected discoveries thatAnnexin A1 and Rab23 levels are elevated in kidney specimens and urinesamples of patients suffering from various kidney disorders as comparedto healthy humans.

Accordingly, one aspect of the present invention features a method ofdiagnosing a renal disorder in a subject (e.g., a human) using eitherAnnexin A1 or Rab23 as a marker. This method includes (i) providing arenal sample (e.g., a kidney tissue sample or a urine sample) obtainedfrom a subject, (ii) detecting the level of Annexin A1 or Rab23 in therenal sample, and (iii) determining whether the subject suffers from oris at risk for a renal disorder (e.g., kidney sclerosis, kidneyfibrosis, glomerular injury, kidney failure, glomerulosclerosis, orglomerulonephritis) based on the Annexin A1 or Rab23 level thusdetected. An elevated level in the subject relative to that in a healthysubject indicates that the subject has or is at risk for the renaldisorder. The level of Annexin A1 or Rab23 can be determined byexamining either its protein or message RNA level. When Rab23 is used asa marker, its level can also be determined by the amount of Shh, Ihh,Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2, Gli3, or a combination thereof.

Another aspect of this invention features a method of assessing theefficacy of a renal disease treatment in a patient. This method includes(i) detecting the level of either Annexin A1 or Rab23 in the kidney of apatient before and after the treatment, and (ii) determining efficacy ofthe treatment based on a change of the Annexin A1 or Rab23 level afterthe treatment. A decreased level of either indicates that the treatmentis effective in that patient.

Also within the scope of this invention is use of Annexin A1 or Rab23 asa biomarker for diagnosing a kidney disease and assessing efficacy of akidney disease treatment, and for manufacturing kits used for thejust-mentioned purposes.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several examples, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first described.

FIG. 1 is a chart showing the protein levels of Annexin A1 in urinesamples obtained from patents having various kidney diseases.

FIG. 2 is a diagram showing that focal segmental glomerulosclerosis(FSGS) progresses over time in a mouse model. Panel A: a chart showingincrease of urine protein levels over time. Panel B: a chart showingincrease of serum creatinine levels over time. Panel C: a chart showingincrease of blood urea nitrogen levels over time.

FIG. 3 is a diagram showing the correlation between the levels of Rab23in kidney tissues and the progress of FSGS in a mouse model. Panel A: achart showing that the protein levels of Rab23 in kidney tissuesincrease over time. Panel B: a chart showing that protein levels ofRab23 in kidney tissues increase over time.

FIG. 4 is a chart showing that the expression levels of hedgehogsignaling pathway genes increase in kidney tissues of FSGS mice overtime.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that the expression levels of Annexin A1 and Rab23are elevated in patients suffering from various kidney diseases ascompared to those in healthy humans. We have also discovered that thelevel of Annexin A1 or Rab23 correlates with disease severity.Accordingly, disclosed herein is a method of using Annexin A1 or Rab23as a biomarker for diagnosing a kidney disease or for assessing theefficacy of a kidney disease treatment in a patient. See Huang et al.,Nephrol Dial. Transplant, 24:743-754 (2009).

Annexin A1, also known as Lipocortin I, is a Ca²⁺-dependentphospholipid-binding protein having a molecular weight of approximately35,000 to 40,000. The amino acid sequence and gene sequence of mouseAnnexin A1 can be found in GenBank (e.g., GenBank accession numbersNP_(—)034860; 17 May 2009 and NM_(—)010730; 17 May 2009, respectively)and the amino acid/gene sequences of human Annexin A1 can be found inGenBank (e.g., GenBank accession numbers AAH35993; 15 Jul. 2006 andBC035993; 15 Jul. 2009).

Rab23 is a small GTPase involved in the hedgehog signaling pathway.Mouse Rab23 is presented in GenBank with accession numbersNM_(—)001153201 (amino acid sequence, 10 May 2009) and NM_(—)008999(gene sequence, 10 May 2009). Human Rab23 can be found in GenBank withaccession numbers BAA87324 (amino acid sequence, 19 Feb. 2008) andAB034244.1 (gene sequence, 19 Feb. 2008).

To diagnose a renal disease by the method of this invention, a renalsample is obtained from a subject who is suspected of having the diseaseand the level of Annexin A1 or Rab23 in the sample is determined. Anelevated level of either Annexin A1 or Rab23 in the renal samplerelative to that of a healthy subject indicates that the subject iseither suffering from the disease or at risk for developing the disease.The term “a renal sample” used herein refers to a biosample that iscommonly used for diagnosing a kidney disease. Examples include, but arenot limited to, a kidney tissue sample and a urine sample. The level ofAnnexin A1 or Rab23 can be determined by examining either its protein ormRNA level via conventional methods. For example, the protein level canbe determined by, e.g., immunohistochemistry, westernblot, SDS-PAGE, or2-dimensional electrophoresis and the mRNA level can be determined by,e.g., real-time RT-PCR.

When Rab23 is used as the marker, its level also can be determined byexamining the protein or mRNA level of one or more other components ofthe hedgehog signaling pathway, e.g., Shh, Ihh, Dhh, PTCH1, PTCH2, SMO,Gli1, Gli2, and Gli3. Table 1 below lists the GenBank accession numbersof these components (mouse and human, referring to the most updatedversions available as of Jun. 15, 2009).

TABLE 1 Human and Mouse Hedgehog Signaling Pathway Components HedgehogSignaling Pathway Components Mouse Human Shh NM_009170 NM_000193 IhhNM_010544 NM_002181 PTCH1 NM_008957 NM_000264 PTCH2 NM_008958 NM_003738SMO NM_176996 NM_005631 Gli1 NM_010296 NM_001160045 Gli2 NM_001081125NM_005270 Gli3 NM_008130 NM_000168

To assess the efficacy of a kidney disease treatment in a patient, renalsamples are obtained from that patient before and after the treatmentand the levels of either Annexin A1 or Rab23 in the samples aredetermined as described above. A decreased level of either Annexin A1 orRab23 after the treatment is indicative of its effectiveness.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference.

Example 1 Correlation Between Annexin A1 and Renal Disease in Humans

Urine samples and kidney tissue samples were collected from 55 patientssuffering from various kidney diseases and 10 healthy volunteers. All ofthe patients and healthy volunteers are Mongolian. The clinical data ofthe patients are shown in Table 2 below:

TABLE 2 Clinical Data of Kidney Disease Patients Clinical features Dailyprotein Renal function Age/ loss SCr BUN CCR Patient Gender Diagnosis(mg/day) (mg/dl) (mg/dl) (ml/min) 1 75/ Crescentic GN 7.6 56 2 47/Chronic GN 1242 14.5 112 3 55/ Focal necrotizing 2550 1.4 21glomerulonephritis with crescentic and global glomerulosclerosis 4 29/End-stage renal disease 1092 10.7 108 5 31/ End-stage renal disease 10928.1 76 4.9 6 58/ Interstitial nephritis with 1235 2.7 56 17.96 focalglomerular sclerosis 7 41/ c/w IgAN with global 1.4 16 sclerosis 8 61/MGN with focal global 2862 0.4 18 120.13 sclerosis 9 38/ c/w Beroganephrosclerosis 1670 7.4 103 15.79 10 35/ Global sclerosis 18644 0.8 21133.93 11 23/ MsPGN with glomerulosclerosis 2414 0.9 14 and interstitialnephritis 12 68/ Interstitial nephritis with 4.1 70 15.65 focalglomerular sclerosis 13 54/ Glomerulosclerosis 3528 4.8 47 18.33 14 75/c/w FSGS in chroni GN stage 95 3.2 45 29.11 15 77/ FSGS 1.7 25 16 21/FSGS 225.5 0.9 10 89.85 17 52/ FSGS 4576 0.9 31 50.7 18 51/ c/w Diabeticnephropathy 23276 10 63 19 42/ c/w Diabetic nephropathy 10.4 101 20 65/c/w Diabetic nephropathy 6741 1.1 24 49.84 21 39/ c/w Diabeticnephropathy 0.7 15 22 69/ c/w Diabetic nephropathy 7079 0.5 18 23 15/IgAN 0.6 16 24 24/ IgAN 1.2 11 25 31/ IgAN 0.8 17 26 29/ IgAN 19 27 28/Lupus nephritis 4c 5670 0.6 11 28 34/ Lupus nephritis 4c 3744 4.2 66 2934/ Lupus nephritis 4c 4587 11.2 126 30 44/ Lupus nephritis 4c 1127 0.617 31 27/ Lupus nephritis 4c 2430 1.6 36 32 25/ Lupus nephritis 4c 19609 53 33 87/ MGN 3.8 44 34 46/ MGN 0.7 20 35 40/ MGN 0.7 7 36 74/ MGN9295 2.6 55 11.75 37 26/ Chronic GN with interstitial 1569 12.4 109 17.1nephritis 38 78/ Focal segmental necrotizing 9000 2.4 75 20.87 GN withinterstitial nephritis 39 60/ Interstitial nephritis 1726 3.1 56 24.5240 59/ Chronic GN with interstitial 1200 7.6 85 4.09 nephritis 41 53/Interstitial nephritis 1.1 17 42 55/ Chronic glomerulonephritis 4.1 64with interstitial nephritis 43 70/ Chronic GN with interstitial 3.7 6918.66 nephritis 44 19/ Chronic interstitial 3604 16.9 132 1.15 nephritis45 25/ proliferation GN 1309 1 10 33.35 46 47/ Chronic GN 1240 25.4 1870.77 47 53/ Chronic GN 3.6 39 48 67/ MPGN 1.6 33 30.31 49 28/ Glomerularfocal hyalinosis 12925 1.1 11 150.51 50 27/ c/w ATN 1700 1.6 80 50.99 5123/ MCD 15190 0.9 11 52 29/ MCD 0.9 13 53 25/ MCD 1665 1 15 138.33 5425/ MCD 2451 1 16 106 55 22/ MCD 90 0.7 8 73.2

The Annexin A1 levels in these samples were examined as follows.

First, an immunohistochemical staining (IHC) assay was performed todetect the protein levels of Annexin A1 in kidney tissue samples.Briefly, the kidney tissue samples were formalin-fixed andparaffin-embedded following routine procedures. After removal ofparaffin, the samples were heated in a microwave for 10 mins (forantigen retrieval) and then cooled to 25° C. for 20 minutes. Afterwards,the samples were immersed in 5% bovine serum albumin to reduce stainingbackground. The samples were then incubated with an anti-human AnnexinA1 antibody (BD biosciences) at 4° C. overnight. After being washedseveral times to remove unbound antibodies, the samples were firstincubated with a biotinylated anti-human IgG antibody and then withreactants for signal development. The staining patterns of the sampleswere observed using a microscope. Results obtained from this assayindicate that the protein levels of Annexin A1 are significantly higherin the kidney tissue samples obtained from the patients than in thekidney tissue samples obtained from the volunteers.

Next, an in-situ hybridization (ISH) assay was performed to examine themessage RNA (mRNA) levels of Annexin A1 in tissue samples obtained fromboth the patients and the volunteers. The following primers were used toproduce a DNA fragment encoding human Annexin A1: forward primer:5′-TTGAGGAGGTTGTTTTAGCTCTG-3′ (SEQ ID NO.: 1); reverse primer:5′-AGTTCTTGATGCCAAAATCTCAA-3′ (SEQ ID NO.: 2). The PCR product wascloned into pGEM-T EASY vector (Promega, Wis., USA) and in vitrotranscription was performed to produce a digoxigenin-labeled RNA probe,which is complementary to the mRNA of Annexin A1. This probe was used tohybridize with the tissue samples mentioned above. Briefly, the tissuesamples were formalin-fixed and paraffin-embedded following routineprocedures. After removal of paraffin, the samples were treated 20 μg/mlProteinase K (Sigma, Mo., USA). They were then hybridized with thedigoxigenin-labeled RNA probe mentioned above in a hybridizationsolution containing 2 mmol/L ethylenediaminetetraacetic acid (EDTA), 20mmol/L Tris, pH7.5, 0.6 mol/L NaCl, 2×Denhardt's solution, 20% dextransulfate, 0.1 mg/mL tRNA, and 0.2 mol/L DTT. The hybridization reactionwas carried out at 42° C. for 16 hrs. After being washed, the sampleswere soaked in 1× blocking solution (Roche, Ind., USA) and incubatedwith an anti-digoxigenin antibody conjugated with alkaline phosphatasefor 1 hour. Substrate NBT/BCIP was then added for color development. Thesamples were further incubated with methyl green (for nuclear staining)and then observed using a microscope. Results thus obtained show thatthe levels of Annexin A1 mRNA are high in kidney tissues obtained fromthe patients, particularly in glomerular endothelial cells, mesangialcells, and damaged kidney areas (e.g., glomerular fibrosis areaes, areaswhere inflammatory cells infiltrated). Differently, the Annexin A1 mRNAlevels in healthy volunteers are much lower. These results areconsistent with those obtained from the IHC assay described above.

Finally, the protein levels of Annexin A1 in urine samples collectedfrom the 55 patients and 10 healthy volunteers mentioned above wereexamined by westernblot as follows. The urine samples were centrifugedat 1000 g for 10 mins. If necessary, the supernants were mixed with 1%antiproteinase (Roche, Ind., USA) and stored at −70° C. for furtheranalysis. The supernatants were mixed with 1× sample buffer (10% SDS, 2ml glycerol, 100 mM DTT, 145 mg Tris, and 0.05% bromophenol blue) andincubated on ice for 15-20 minutes. The proteins contained in themixtures were separated by SDS-Polyacrylamide Gel Eletrophoresis(SDS-PAGE; 10%) and transferred to a nitrocellulose membrane. Themembrane was treated with a blocking buffer (5% non-fat milk) for 2 hrs,incubated with an anti-human AnxA1 antibody (BD biosciences) at 4° C.overnight, and then incubated with a horseradish peroxidase-conjugatedanti-human IgG antibody at room temperature for 1 hr. The membrane waswashed several times and incubated with a chemiluminescent reagent(PerkinElmer Life Sciences, Boston, Mass.) for signal development. Theresults thus obtained were normalized against the creatine levels in thesame urine samples. Annexin A1 protein was detected in urine samples ofpatients with various renal disorders. As shown in FIG. 1, the amountsof Annexin A1 protein in the patient urine samples are significantlyhigher than those in the healthy volunteers.

Taken together, the results discussed above indicate that Annexin A1 isa biomarker associated with kidney disease.

Example 2 Correlation Between Rab23 and Focal SegmentalGlomerulosclerosis in Mice

8-week-old BALB/c mice were injected with adriamycin (0.1 mg/per 10 gbody weight) intravenously to induce Focal Segmental Glomerulosclerosis(FSGS). Blood, urine, and kidney tissue samples were collected at days0, 7, 15, and 20 after injection. More specifically, the blood sampleswere collected from the retro-orbital venous plexus and the urinesamples were collected by gentle abdominal massage. The samples werecentrifuged to remove insoluble substances and then stored in liquidnitrogen. When urine proteins are to be measured, the correspondingsamples were collected on parafilms. The modified Bradford protein assaywas performed to examine urine protein concentration, the picric acidcolorimetric kit (Sigma 555-1) was used for detecting creatinine, and anurease assay kit provided by Sigma (640-5) was used for determining thelevel of blood urea nitrogen (BUN).

Adriamycin treatment resulted in severe propeinuria and reduced renalclearance function. Compared with the control mice, the mice treatedwith adriamycin showed substantially increased urine protein levels onday 15 (i.e., 0.59±0.082 versus 16.84±3.13 mg/ml, F=13.81, P<0.01). SeeFIG. 2A. The levels of serum creatinine and BUN, two common parametersfor evaluating renal functions, are also higher in theadriamycin-treated mice than in the control mice. More specifically, onday 15, the serum creatinine level in treated mice is 2.02±0.17 mg/dl,much higher than that in the control mice, i.e., 0.48±0.03 mg/dl(F=25.17, p<0.01) and the BUN level in treated mice is 71.42±8.09 mg/dl,also much higher than that in the control mice, i.e., 21.85±1.99 mg/dl(F=38.79, p<0.01). See FIGS. 2B and 2C.

The protein levels of Rab23 in the urine samples were analyzed by a2-dimentional isoelectro focusing (IEF)/SDS-PAGE assay. The urinesamples obtained from both FSGS mice and control mice were subjected toultrafiltration, desalination and concentration. They were then loadedonto an Immobiline DryStrip gel (3-10, GE Healthcare, N.J., USA) withimmobilized pH gradient (IPG) for simultaneous rehydration. After beingseparated by IEF, the proteins were subjected to SDS-PAGE analysis. Theprotein migrated to the position corresponding to Rab23 was eluted anddigested with trypsin. The resultant peptides were analyzed by massspectrometry, using Bruker Biflex IV MADLI-TOF MS (Bruker Daltonics,Bremen, Germany). Peptide mass fingerprinting (PMF) was obtained basedon the signals generated by 500 laser shots. The data thus obtained wereanalyzed using Flexanalysis™ and BioTools™ software (Bruker Daltonics,Bremen, Germany). By searching against the UniProt database(http://www.pir.uniprot.org) with the MS-Fit database searching engine,the amino acid sequences of the peptides were obtained, which match 10Rab23 fragments. In this assay, Rab23 was detected in the urine samplesobtained from the FSGS mice but not the control mice, indicating thatthe expression of this protein is only detectable in mice having kidneydisease.

The protein levels of Rab23 were also examined by westernblot. 15 μl ofeach urine sample, 15 μl of each blood sample, 50 μg kidney proteinsextracted from each tissue sample, and 50 μg proteins extracted fromcultured rat mesangial cells were separated by SDS-PAGE (10%). Theproteins were then transferred to a Hybond PVDF membrane (GE Healthcare,N.J., USA). The membrane was blocked in 20 ml blocking buffer(Tris-buffered saline, pH 8.0, containing 0.05% Tween-20 (TBST) and 5%skimmed milk) at room temperature for 1 hr, incubated with a rabbitanti-Rab23 antibody (1:1000) at room temperature for 1 hr, washed threetimes with TBST, and incubated with a goat anti-rabbit IgG antibody atroom temperature for 1 hr. An ECL kit was used for color development.The intensity of the color was quantified with a densitometer. The datathus obtained were normalized against the levels of GAPDH in the samesamples. Rab23 protein was detected in the urine samples of FSGS micecollected on days 7, 15, and 20. The levels of Rab23 protein weresignificantly increased on days 15 and 20 as compared to that on day 7.See FIG. 3A.

Next, kidney tissue samples obtained from both FSGS mice and controlmice were subjected to immunostaining. The tissue samples, obtained ondays 0, 7, 15, and 20 after injection of adriamycin, were OCT-embeddedand cut into sections having a thickness of 5-μm. The sections wereimmersed in acetone for 5 mins. After being air dried, the sections werefirst incubated in a Tris buffered saline (TBS, pH=7.4) containing 0.05%Tween 20 (TBST) and 2% bovine serum albumin at room temperature for 30mins, then with a rabbit anti-mouse Rab23 (1:200 dilution) antibodyalone, or with both the anti-Rab23 antibody (1:150) and a goatanti-nephrin (1:20) (Santa Cruz) at 4° C. overnight. Nephrin is a markerof mesangial cells. After being washed with TBST, the sections wereincubated with a horseradish peroxidease (HRP)-conjudated goatanti-rabbit IgG antibody (1:200) (Jackson ImmunoResearch, West Grove,Pa.) or a combination of fluorescein isothiocyanate (FITC)-conjugateddonkey anti-rabbit IgG antibody (1:500) (Jackson ImmunoResearch, WestGrove, Pa.) and Alexa Fluor 594-conjugated donkey anti-goat IgG antibody(1:500)(Invitrogen, Calif., USA) at room temperature for 1 hr. Substrate3,3′-diaminobenzidine (DAKO, Carpinteria, Calif.) was used for signaldevelopment. The sections were also counterstained with hematoxylin. Theintensity of the signals released from each section was semi-quantifiedunder an optical microscope. Briefly, 50 glomeruli were examined on eachslide and a score ranging from 0 to 3 was assigned to glomeruli,podocytes and mesangial cells based on the signal intensities thereof.The score of the total intensity was calculated as follows:Total intensity score=[(negative intensity) %×0]+[(very weak intensity)%×0.5]+[(intensity score being 1)%×1]+[(intensity score being2)%×2]+[(intensity score being 3)%×3)].

As compared with the control mice, the FSGS mice showed a much higherexpression levels of Rab23, particularly in mesangial cells. Theintensity scores of the mesangial cells in the FSGS mice at days 7, 15,and 20 are 145.33±17.83, 189.33±21.63, and 219.17±24.78, respectively,which are much higher than the mean intensity score of the control mice,i.e., 67.33±4.72. The protein levels of Rab23 in the kidney tissuesamples of the FSGS mice also increased along with disease progress. Atday 7, the intensity score representing the Rab23 protein level is0.61±0.078 while at days 15 and 20, this score increased to 1.02±0.11and 0.93±0.090, respectively (p<0.01). These results indicate that theexpression level of Rab23 correlates to disease severity.

The levels of Rab23 in the kidney tissues of the FSGS mice and controlmice were further analyzed by examining its mRNA levels. Total RNAs wereextracted from kidney tissues using the Trizol reagent (LifeTechnologies, Rockville, Md.). 3 μg of the total RNAs were mixed with200 ng random primers, 0.5 mM dNTPs, 1× first stand buffer, 5 mM DTT, 10units of reverse transcriptase (Invitrogen, Calif., USA) in a totalvolume of 20 μl for EDNA synthesis via reverse transcription (RT). ThecDNA products were subjected to polymerase chain reaction as follows. 10μl of the RT reaction mixture was mixed with 0.1 μM of the primers5′-GAAGGAGGACC TCAACGTGAGTGA-3′ (forward primer SEQ ID NO.: 3) and5′-CCAAGTGACTTCTGACCGATGCA-3′ (reverse primer, SEQ ID NO.: 4), 1×PCRbuffer, 50 μM dNTPs, 1 unit of KlenTag DNA polymerase, and 12.5 μlBio-Rad iQ SYBR Green supermix in a total volume of 25 μl, using theCycler real-time PCR instrument provided by Bio-Rad. The PCR reactionwas carried out under the following conditions:

initial denaturation and enzyme activation: 95° C. for 15 mins;

amplification: 94° C. for 15 s, 55° C. for 35 s, and 72° C. for 60 s, 50cycles; and

final elongation: 72° C. for 10 mins.

The amounts of the PCR products were quantified, normalized against theamounts of GAPDH mRNA in the same samples using a comparative thresholdcycle (2-^([delta]Ct)) method. The data thus obtained, expressed asmeans± standard errors of the means (SEM), were analyzed by one-wayanalysis of variance (ANOVA) using the SPSS software. The Newman-Keulstest was performed to determine the p-values of the ANOVA results.P<0.05 indicates that the results are statistically significant.

In kidney tissue samples obtained from the FSGS mice at different timepoints, the Rab23 mRNA levels at days 7, 15, and 20 were 0.12±0.019,0.18±0.028, and 0.27±0.059, respectively. See FIG. 3B. These levels aremuch higher than the Rab23 mRNA level in the control mice, i.e.,0.013±0.0025 (p<0.01). This result indicates that the Rab23 levelscorrelate with FSGS severity.

The mRNA level of Rab23 was also examined in mouse mesangial cell lineCRL-1927, following the same procedures described above. The resultsindicate that Rab23 is expressed at high levels in mesangial cells.

Example 3 Correlation Between Levels of Rab23 and Kidney Disease inHumans

The Rab23 levels in the kidney tissue samples as described above,obtained from the 55 human patients and 10 healthy volunteers, werefirst examined by immunostaining. The tissues were cut into sections.The sections were first incubated in TBS (Tris buffered saline, pH=7.4)containing 0.05% Tween 20 (TBST) and 2% bovine serum albumin at roomtemperature for 30 mins, and then incubated with a rabbit anti-humanRab23 antibody (1:200 dilution) at 4° C. overnight. After being washedwith TBST, the sections were further incubated with a horseradishperoxidease (HRP)-conjugated goat anti-rabbit IgG antibody (1:200)(Jackson ImmunoResearch, West Grove, Pa.) at room temperature for 1 hr.Substrate 3,3′-diaminobenzidine (DAKO, Carpinteria, Calif.) was thenadded for signal development. The sections were also counterstained withhematoxylin. The intensities of the signals were semi-quantitated usingan optical microscope. Rab23 was found to be expressed at high levels inpodocytes and wall cells of glomeruli in the kidney tissue samplesobtained from the patients, while only very lower levels of Rab23 weredetected in the kidney tissue samples of the healthy volunteers.

The Rab23 levels in the urine samples obtained from both the humanpatients and volunteers were further determined by western blotfollowing the procedures described in Example 2 above. The proteinlevels as shown in the westernblot were quantified and normalizedagainst the creatinine concentrations in the same urine samples. Resultsthus obtained indicate that the levels of Rab23 in the patient urinesamples are higher than those in the urine samples of the healthyvolunteers.

Finally, the Rab23 levels in the kidney tissue samples were examined byreal-time RT-PCR, following the procedures described in Example 2 above.The results are consistent with those described above.

Taken together, this study demonstrates that Rab23 is a biomarker ofkidney disease.

Example 4 Determining Levels Hedgehog Signaling Pathway Components inFSGS Mouse Kidney Tissues

The expression levels of hedgehog signaling pathway genes, i.e., Shh,Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli 2, and Gli3, were determined byreal-time RT-PCR following the method described in Example 2 above. Asshown in FIG. 4, these genes were expressed in kidney tissues of theFSGS mice and their expression levels correlate with disease severity.This result indicates that the level of any of these hedgehog signalingpathway components, or their combination, can be used for kidney diseasediagnosis and prognosis.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

1. A method for diagnosing a renal disorder in the kidney of a subject,comprising: providing a renal sample obtained from a subject suspectedof having a glomerulonephritis or a renal fibrosis; detecting the levelof a biomarker in the renal sample; and determining whether the subjecthas the glomerulonephritis or the renal fibrosis based on the level ofthe biomarker thus detected; wherein the biomarker is Annexin A1 and anelevated level of the biomarker in the subject relative to that in ahealthy subject indicates that the subject has the Annexin A1 whichappears in the kidney tissue sample while the kidney has theglomerulonephritis or the renal fibrosis.
 2. The method of claim 1,wherein the detecting step is performed by examining the protein levelof Annexin A1.
 3. The method of claim 1, wherein the detecting step isperformed by examining the message RNA level of Annexin A1.
 4. Themethod of claim 1, wherein the biomarker is Rab23.
 5. The method ofclaim 4, wherein the detecting step is performed by examining theprotein level of Rab23.
 6. The method of claim 4, wherein the detectingstep is performed by examining the message RNA level of Rab23.
 7. Themethod of claim 4, wherein the detecting step is performed by examiningthe amount of Shh, Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2, Gli3, or acombination thereof, and determining the level of Rab23 based on theamount thus obtained.
 8. The method of claim 4, wherein the renaldisorder is selected from the group consisting of kidney sclerosis,kidney fibrosis, glomerular injury, kidney failure, glomerulosclerosis,and glomerulonephritis.